A distinguishing feature of eukaryotic cells is the presence of an array of intracellular membrane enclosed organelles. This highly compartmentalized organization is essential to the normal functioning of the cell. A variety of specific proteins, lipids, and carbohydrates must be efficiently delivered to these organelles. Misdirection of these molecules can lead to cell and organism malfunction (e.g., I-cell disease). The molecular mechanisms by which proteins are sorted into the different cellular compartments are at present largely unknown. Yeast provide an excellent genetic system for addressing these questions. Yeast and higher eukaryotes appear to contain nearly identical pathways for the maintenance and assembly of these complex cellular structures. In this proposal focus will be put on analyzing the mechanism of specific protein targeting to three yeast subcelluar compartments. Representative proteins will be studied; the secreted proteins invertase and Alpha-factor, the vacuolar carboxypeptidase Y, and the mitochondrial ATPase Beta-subunit. The genes coding for these proteins have all been cloned on yeast plasmids. To facilitate the analysis, gene fusions have been or will be constructed between these genes and either the lacZ gene of E. coli (codes for the enzyme Beta-galactosidase) or the SUC2 gene of yeast (codes for the enzyme invertase). These enzymes provide useful biochemical tags for following the localization of the hybrid proteins encoded by the gene fusions. Analysis of a variety of different size classes of fusions should enable one to dissect a gene into the various component signals that are responsible for the efficient targeting of these molecules to their respective cellular locations. In addition, by exploiting certain phenotypes conferred to the yeast cell by these hybrid proteins, it should be possible to isolate localization defective mutants. Such mutants could define critical recognition signals or specific recrptors that participate in the delivery process. Cloning and characterization of the genes and sequences identified by this mutational analysis will provide a more precise picture of the molecular events involved in the maintenance of cellular organelle structure and function.